Sydowia 67 (2015) 119

DOI 10.12905/0380.sydowia67-2015-0119

Morphology and phylogeny of Acaulospora foveata (Glomeromycetes) from Mexico

Dora Trejo1, Gastón Guzmán2, Liliana Lara1, Ramón Zulueta1, Javier Palenzuela3, Iván Sánchez-Castro4, Gladstone Alves da Silva5, Ewald Sieverding6 & Fritz Oehl5,7,*

1  Laboratorio de Organismos Benéficos, Facultad de Ciencias Agrícolas, Universidad Veracruzana, Gonzalo Aguirre Beltrán S/N, Zona Universitaria, CP 91090, Xalapa, Veracruz, México

2 Instituto de Ecología, A.C., Xalapa, Veracruz, México3 Departamento de Microbiología del Suelo y Sistemas Simbióticos, Estación Experimental del Zaidín, CSIC,

Profesor Albareda 1, E-18008 Granada, Spain4 Departamento de Microbiología, Campus de Fuentenueva, Universidad de Granada, E-18071 Granada, Spain

5 Departamento de Micologia, CCB, Universidade Federal de Pernambuco, Av. da Engenharia s/n, Cidade Universitária, 50740-600, Recife, PE, Brazil

6  Institute of Plant Production and Agroecology in the Tropics and Subtropics, University of Hohenheim, Stuttgart, Germany7  Agroscope, Federal Research Institute for Sustainability Sciences, Plant-Soil-Interactions, Reckenholzstrasse 191,

CH-8046 Zürich, Switzerland

*  e-mail: fritz.oehl@gmail.com

Trejo D., Guzmán G., Lara L., Zulueta R., Palenzuela J., Sánchez-Castro I., Alves da Silva G., Sieverding E. & Oehl F. (2015) Morphology and phylogeny of Acaulospora foveata (Glomeromycetes) from Mexico. – Sydowia 67: 119-126.

Acaulospora foveata, a glomeromycete fungus with pitted ornamentation on the outer spore surface, was originally isolated from a sugar cane field close to the city Orizaba, Veracruz, Mexico, in 1982. At that time, the concepts of morphological spore descriptions were less evolved, and only two wall layers are mentioned in the protologue, an outer pigmented layer and an inner hyaline layer. In recent years, several Acaulospora spp. with pitted ornamented spore surfaces were described. In order to mini-mize errors on the identification of A. foveata and similar fungal species, we studied the holotype, isotype and newly collected epitype material from the type location and morphologically re-described the fungus from these types, and we performed mo-lecular phylogenetic analyses of sequences obtained from the ribosomal gene gained from single spores collected at the type loca-tion. Acaulospora foveata has - like many other species in the genus - three spore walls, including an inner, ‘beaded’ wall, and at least eight spore wall layers. Phylogenetically, the fungus clusters in a clade well separated from all other known Acaulospora spp., and it is most closely related to A. lacunosa. We conclude that with this new information A. foveata can now be correctly identified also by molecular analyses. 

Keywords: Arbuscular mycorrhizal fungus, Glomeromycota, Acaulosporaceae.

Many arbuscular mycorrhizal (AM) fungal spe-cies were described  in the 1970’s and 1980’s, after spores could be isolated by new methods and easier be identified by type of spore formation and spore morphology  (e.g.  Gerdemann  &  Trappe  1974, Schenck et al. 1984). The number of species descrip-tions increased rapidly and a high diversity of AM fungi was virtually found in all  terrestrial ecosys-tems with flowering plants (e.g. Cabello et al. 1994, Oehl & Körner 2014). However, little attention was initially given to exact wall  layer descriptions,  i.e. outer and inner walls and layers of each wall (e.g. Hall 1977, Nicolson & Schenck 1979). Specific no-menclature  for  wall  layers  started  with  detailed wall  and  wall  layer  definitions  and  elaborated ‘murographs’ (e.g. Walker 1983, Schenck et al. 1984). Since then specific wall characteristics and defini-

tions  were  proposed,  and  new  interpretations  are still  being proposed  (e.g. Oehl & Sieverding  2004, Oehl et al. 2008, 2011 a, Lima et al. 2014, Marinho et al. 2014, Sieverding et al. 2014, Błaszkowski et al. 2015). Today it is clear that especially Acaulospora spp. have spores with three walls, of which each can have  multiply  layers  (e.g.  Schenck et  al. 1984, Stürmer & Morton 1999).

Several key species of Acaulosporaceae were al-ready described in the 70th and 80th, e.g. A. laevis, A. elegans, A. scrobiculata, A. spinosa and A. fovea-ta (Gerdemann & Trappe 1974, Trappe 1977, Walker & Trappe 1981, Janos & Trappe 1982). While some of them have more or less clear diagnostic ornamenta-tions on the outer spore surface, the morphological identification of others has became uncertain, since later other species with similar wall characteristics 

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have  been  separated,  those  usually  based  on  con-comitant morphological and molecular phylogenet-ic analyses in the more recent descriptions. We see the need to isolate again the earlier described Acau-lospora spp. from the locations of the types, to re-describe  the  species  on  the  base  of  new  observa-tions, and to generate molecular phylogenetic data for  those species. We especially want to more pre-cisely delimit the surface ornamentation structures, and  the wall  and wall  layer  composition,  as  such morphological  characteristics  are  often  the  first step  to  identify  and  organize  species  in  diversity studies.

The objective of this study was therefore to re-isolate A. foveata Trappe & Janos (Janos & Trappe 1982)  from  its  type  location  in Orizaba  (Veracruz State) of tropical Mexico, to re-examine the spores of the type, to complement the species description, and to extract and amplify DNA from the spores for subsequent molecular phylogenetic analyses on the ribosomal gene. 

Material and methods

Study site 

The  type  location was  briefly  described  in  the protologue  as  a  sugar  cane  field  ‘approximately 5 km Northeast of the city Orizaba’ (Janos & Trappe 1982). We took soil samples (about 1 kg) at about the same  distance  and  orientation  from  the  city,  and also in a sugar cane field (18° 53’ 30’’ N, 97° 02’ 30’’ W, about 1150 m a.s.l.), in November 5, 2013. The site is located in the central mountainous zone of the Si-erra Madre oriental, next to the valley of the volca-no Orizaba (5636 m a.s.l.). The soil type is a clayey Andosol with pH of  approximately  4.0. The mean annual temperature at study site is about 25°C, and the mean annual rainfall is circa 2700 mm. The nat-ural vegetation in the surroundings of the study site is an evergreen tropical rainforest.

Morphological analyses

Holotypes and isotypes of A. foveata, originally deposited  by  Janos  & Trappe  at  the  Mycological Herbarium of the Oregon State University (OSC, in Corvallis, Oregon, USA), at the Escuela Nacional de Ciencias Biológicas (ENCB, in Mexico City, Mexico) and at  the  Instituto de Ecología  (XAL)  in Xalapa (Veracruz, Mexico) were re-analyzed as described in preceding publications (e.g. Oehl et al. 2008, 2011 a, 2011 b).

Spores  from  the  newly  collected  soils,  derived from the type area in ‘approximately 5 km North-

east  of  the  city  Orizaba’  (Janos  &  Trappe  1982), were  isolated  and  analyzed,  as  described  in Sieverding  (1991). They  were  analyzed  in  polyvi-nyl  alcohol-lactic  acid-glycerol  (PVLG;  Koske  & Tessier  1983),  in  a mixture  of PVLG and Melzer’s reagent (Brundrett et  al. 1994),  a mixture  of  lac-tic  acid  to  water  at  1:1,  Melzer’s  reagent,  and  in water  (Spain  1990). The  terminology  of  the  spore wall  structure  basically  is  that  presented  in  Oehl et al. (2012)  for Acaulosporaceae. Newly prepared slides  were  deposited  as  epitypes  at  the  Myco-logical  Herbarium  of  ETH  Zurich  (Z+ZT),  at  the Instituto  de  Ecología  (XAL)  and  at  the Universi-dad  Veracruzana  (Laboratorio  de  Organismos Benéficos). 

Molecular analyses

Spores were  isolated  from  the  soil  taken  from the type location in November 2013. They were sur-face-sterilized with chloramine T (2 %) and strepto-mycin  (0.02  %)  (Mosse  1962)  and  crushed  singly with a sterile disposable micropestle in 40 µl milli-Q water, as described in Palenzuela et  al. (2013). PCRs using the crude extracts as target were per-formed in an automated thermal cycler (Gene Amp PCR System 2400, Perkin-Elmer, Foster City, Cali-fornia)  with  a  pureTaq  Ready-To-Go  PCR  Bead (Amersham Biosciences  Europe GmbH, Germany) following manufacturer’s instructions, with 0.4 µM concentration of each primer. A two-step PCR am-plified  the  SSU  end,  ITS1,  5.8S,  ITS2  and  partial LSU rDNA fragment using the SSUmAf/LSUmAr and  SSUmCf/LSUmBr  primers  consecutively (Krüger et al. 2009). PCR products were checked by electrophoresis  in 1.2 % agarose gels stained with Gel Red™ (Biotium Inc., Hayward, CA, U.S.A.) and viewed by UV  illumination. The  amplicons  of  ap-propriate  size  were  purified  using  the  GFX  PCR DNA kit and Gel Band Purification Illustra, cloned into the PCR 2.1 vector  (Invitrogen, Carlsbad, CA, USA),  and  transformed  into  One  shot® TOP10 chemically  competent Escherichia coli  cells. After plasmid isolation from transformed cells, the cloned DNA fragments were sequenced with universal for-ward and reverse vector primers (T7 and M13r) on an  automated DNA  sequencer  (Perkin-Elmer ABI Prism 373). Sequence data were compared to public sequence  databases  using  BLAST  (Altschul et  al. 1990). The percentage of identity among the Acau-lospora sequences was calculated using the BLASTn analysis. The new sequences were deposited in the EMBL  database  under  the  accession  numbers LN736022-LN736027.

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Figs. 1–7. Acaulospora foveata. 1–5. Intact and crushed spores with three multiple-layered walls: OWL1-3, MWL1-2, and IWL1-3. IWL1 with granular, ‘beaded’ structure. In dented and crushed spores (Figs. 2-4), the surface pits appear often more irregular than in totally globose spores (Fig. 1). 5. Spore wall composition; granular structures of IWL1 disappeared through repeated pressure on the cover slide. 6. Crushed spore segment exposed to Melzer’s reagent: IWL2 & IWL3 staining purple to dark purple.

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Phylogenetic analyses

Some Acaulospora  spp.  have  sequences  either from LSU rDNA or from ITS regions (e.g. A. collic-ulosa, A. colossica, A. denticulata, A. dilatata, A. herrerae, A. longula and A. tuberculata). Thus, the phylogeny was reconstructed by independent anal-yses of the ITS region and of the partial LSU rDNA. The  AM  fungal  sequences  obtained  were  aligned with  other Acaulosporaceae  sequences  from Gen-Bank in ClustalX (Larkin et al. 2007). Claroideoglo-mus etunicatum (W.N.Becker & Gerd.) C.Walker & A.Schüssler was included as outgroup. Prior to phy-logenetic analysis, the model of nucleotide substitu-tion  was  estimated  using Topali  2.5  (Milne et  al. 2004). Bayesian (two runs over 2 × 106 generations with a sample frequency of 200 and a burn-in value of 25 %) and maximum likelihood (1000 bootstrap) analyses  were  performed  in  MrBayes  3.1.2  (Ron-quist & Huelsenbeck 2003) and PhyML (Guindon & Gascuel  2003),  respectively,  launched  from Topali 2.5, using the GTR + G model. Maximum parsimony analysis  was  performed  using  PAUP*4b10  (Swof-ford 2003) with 1000 bootstrap replications.

Results

Taxonomy

Acaulospora foveata Janos & Trappe – Figs. 1–7Mycobank MB109576

E p i t y p e .  –  Mexico,  Veracruz,  Orizaba (18°53’30’’N,  97°02’30’’W).  Isolated  from  a  sugar cane  field.  Soil  collected  by Da. H.  Lee Espinoza. Epitype isolated by F. Oehl, L. Capistrán & R. Zu-lueta,  deposited  at  Z+ZT  under  the  accession  ZT Myc 56320, and here designated.

D e s c r i p t i o n .  – Spores  formed  laterally  on the neck of  sporiferous  saccules  in approximately 100–250 µm distance to the saccule terminus. They are yellowish brown to yellow brown to sometimes reddish  brown,  becoming  brown  to  black  brown with age, globose to ellipsoid, 185–310(410) × 215–350(480) µm in diam., and have three walls  (outer, middle, inner wall). The sporiferous saccule termini are of similar size as the spores formed on the sac-cule necks.

Outer wall with  three  layers  (OWL1-3).  OWL1 hyaline to subhyaline, evanescent, 1.0–1.8 µm thick. OWL2 yellowish brown to reddish brown, laminate, 8.5–15 µm thick, with round to oblong, and concave depressions, generally 3.5–8.5(12) × 3.5–12.5(16) µm in diameter,  and 1.5-3.5 µm deep. Regularly,  some pits were also of smaller sizes (up to 1.3 x 1.3 µm). 

OWL3 concolorous with OWL2, and regularly tight-ly adherent to it, persistent and 0.9-1.5 µm thick.

Middle wall with  two almost  identical, flexible and hyaline  layers  (MWL1-2),  together 1.7–2.7 µm thick. 

Inner wall with three layers (IWL1-3). IWL1 hy-aline  to  subhyaline,  1.3–2.4(3.0)  µm  thick,  with granular,  ‘beaded’  structure,  that  may  completely disappear,  and  become  completely  hyaline  under pressure  applied  on  the  cover  slide  in  harshly crushed spores.  IWL2 is hyaline, 2.5–5.5 µm thick, expanding  up  to  15  µm  under  pressure  applied, staining purple to dark purple in Melzer’s reagent. IWL3 also hyaline, 0.6–1.2 µm thick, often showing several  folds  in  crushed  spores,  and  also  staining purple to dark purple in Melzer’s reagent. 

Cicatrix  at  spore  base  closed  by  laminae  of OWL2 and by adherent OWL3, 18-25 µm in diame-ter.

M o l e c u l a r   p hy l o g e n e t i c   a n a l y s e s . – In  the molecular phylogenetic  analyses A. foveata and A. lacunosa formed together a well supported clade  separate  from  other  Acaulospora  spp. (Figs. 8–9). Only one rDNA sequence of A. foveata was  found  in  the NCBI  data  bank  (isolate CR315 from INVAM originating from Costa Rica). The se-quences obtained in the present study grouped with the isolate CR315 in the LSU rDNA tree. It was not possible to separate A. foveata from A. lacunosa in the  LSU  rDNA  tree  (Fig.  8).  However,  the  phylo-gram obtained by the ITS sequences showed A. fo-veata and A. lacunosa in different clades with high support values (Fig. 9). The species most closely re-lated to A. foveata was A. lacunosa with 96 % and 94  %  of  identity  to  the  LSU  rDNA  and  ITS  se-quences, respectively.

The  environmental LSU  rDNA  sequences with closest match  (97–99 %)  to A. foveata were found in roots of mangrove plants (HM570008, HQ243214) sampled  in  Zhuhai  Mangrove  Area,  Guangdong province – China (Wang et al. 2011), roots of Voyr-ia aphylla  (HQ857180-HQ857181)  and  spores (JF276256-JF276257)  collected  in  a  tropical  rain-forest of the Guadeloupe Caribbean Island (Courty et al. 2011) and in soil (JN890255) from a tropical rainforest in Guyana (McGuire et al. 2010). For the ITS region, 98 % of identity was found among se-quences  from A. foveata and those from roots of mangrove  plants  (HM570008, HQ243214)  sampled in Zhuhai Mangrove Area, Guangdong province  – China  (Wang et  al. 2011) and roots of Voyria aphylla  (HQ857180),  collected  in  a  tropical  rain-forest of the Guadeloupe Caribbean Island (Courty et al. 2011).

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Fig. 8. Phylogenetic tree of the Acaulosporaceae obtained by analysis from partial LSU rDNA sequences of different Acaulospora spp. Sequences are  labeled with their database accession numbers. Support values  (from top) are from maximum parsimony (MP), maximum likelihood (ML) and Bayesian analyses, respectively. Topology of the tree is the same presented by the consensus tree of the ML analysis. Sequences obtained in this study are in boldface. Only support values of at least 50 % are shown. Thick branches represent clades with more than 90 % of support in all analyses. The tree was rooted by Claroideoglomus etunicatum. (Consistency Index = 0.50; Retention Index = 0.86).

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Fig. 9. Phylogenetic tree of the Acaulosporaceae obtained by analysis from ITS1, 5.8S rDNA and ITS2 sequences of different Acaulospora spp. Sequences are labeled with their database accession numbers. Support values (from top) are from maximum parsimony (MP), maximum likelihood (ML) and Bayesian analyses, respectively. Topology of the tree is the same presented by the consensus tree of the ML analysis. Sequences obtained in this study are in boldface. Only support values of at least 50 % are shown. Thick branches represent clades with more than 90 % of support in all analyses. The tree was rooted by Claroideoglomus etunicatum. (Consistency Index = 0.49; Retention Index = 0.84).

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Discussion

Acaulospora foveata  was  originally  described with two wall layers (Janos & Trappe 1982). At that time,  the  species  could  easily  be  identified  by  its acaulosporoid  spore  formation  and  the  diagnostic surface ornamentation. It was distinct from those of all other Acaulospora  species with pitted surfaces described until then (A. srobiculata, A. bireticulata and A. elegans; Gerdemann & Trappe 1974; Trappe 1977,  Rothwell  & Trappe  1979).  Five  genera  with acaulosporoid  spore  formation  (sensu  lato),  have hitherto been known: Acaulospora, Otospora, Amb-ispora, Archaeospora and Palaeospora (Spain et al. 2006, Palenzuela 2008, Oehl et  al. 2011  b,  2015). They can clearly be differentiated from each other by spore morphology and especially by spore wall composition (Oehl et al. 2015). 

In this study, it was re-inforced that A. foveata isolated from the type location in Orizaba (Mexico) belongs to the Acaulosporaceae. It has the charac-teristic spore wall composition of this family con-sisting  of  three  spore walls  including  the ‘beaded’ structure of the inner wall surface. The large spore size and the characteristic pitted spore surface or-namentation  makes  it  easy  to  identify A. foveata morphologically,  and  to  group  it  into  the  genus Acaulospora as it was suggested by Janos & Trappe (1982). Acaulospora foveata can be differentiated from A. lacunosa (Morton 1986) and all other known ‘pitted’  Acaulospora  spp.  by  having  substantially bigger spores as summarized in an overview for all Acaulospora spp. in recent published identification keys (Błaszkowski 2012, Oehl et al. 2012).

The ITS and partial sequence of the LSU rDNA analyzed also unequivocally confirmed the correct-ness of the genus and species identification of Janos & Trappe (1982). The fungus is most closely related to A. lacunosa.  In addition,  it was recognized that the A. foveata sequences obtained from spores from Costa  Rica  coincided  with  our  sequences  from Orizaba, Mexico. Our sequences obtained from the location  of  the  type  can  be  used  in  the  future  to identify this species phylogenetically through mo-lecular analyses, globally and  in whatever ecosys-tem.

Acknowledgements

Dra. Hilda E. Lee Espinosa (Universidad Verac-ruzana, Facultad  de  Ciencias  Biológicas  y  Agro-pecuarias-Córdoba, Veracruz, Mexico)  is  acknowl-edged  for  providing  soil  samples  from  the  type location  area  in  Orizaba.  This  study  was  finan-cially  supported  by  the  Apoyo  al  Fortalecimento 

de Cuerpos Académicos  (2013) of  the Universidad Veracruzana, providing a grant to F. Oehl as ‘visit-ing  scientist’. 

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(Manuscript accepted 12 May 2015; Corresponding Editor: I. Krisai-Greilhuber)